The long term goal of this project is to understand the molecular mechanisms responsible for altered glucose homeostasis during highly active antiretroviral therapy (HAART). The metabolic changes that occur in association with the use of HIV protease inhibitors (Pis) have significant potential for increasing cardiac- associated morbidity and mortality. The immediate objectives of this proposal are to determine the specific contribution of GLUT4 inhibition to altered glucose uptake into insulin responsive tissues, to elucidate the role of altered glucose transport in the development of changes in adiocytokine production, and to identify the precise molecular interactions that mediate PI binding to GLUT4. The role of GLUT4 in Pi-induced effects on peripheral glucose disposal will be studied by measuring radiolabeled 2-deoxyglucose uptake into cardiac, skeletal muscle and adipose tissue under basal and hyperinsulinemic euglycemic clamp conditions in wild-type and transgenic mice with specific deletion of GLUT4 from muscle or fat. Changes in leptin, adiponectin and resistin RNA and protein levels, peripheral glucose disposal, and hepatic glucose production will be determined following semi-acute exposure of control and GLUT4 null mice to Pis in the presence and absence of semi-chronic treatment with NRTIs. The structural features in GLUT4 that mediate HIV protease inhibitor binding and inactivation will be determined in primary and/or cultured rat adipocytes labeled with a photoactivatable peptide containing the GLUT4 inhibiting epitope zHFF coupled to the FLAG epitope. Modified proteins will be isolated using a FLAG antibody affinity column and identified by mass spectrometry. The ability of Pis to influence the binding of proteins known to interact with the GLUT4 carboxy terminus will also be investigated by Western blot analysis and co-immunoprecipitation experiments using factor specific antibodies. Mutations will be introduced into the identified GLUT4 PI binding site and the resulting functional effects on transport activity and PI sensitivity will be determined. Taken together, these studies will provide crucial information about the mechanism(s) by which Pis contribute to the development of insulin resistance and diabetes mellitus. This will greatly assist efforts to develop effective strategies for preventing and/or treating the metabolic changes that occur both in patients on HAART and in the wider context of HIV-negative individuals with features of the metabolic syndrome.